The present invention relates to proteins for novel ATP-sensitive potassium channels, huKATP-1 and ruKATP-1, that are expressed in various tissues of human and rat origins, and to genes encoding the same. The said proteins and genes can be used as diagnostic and therapeutic agents for potassium-channel related diseases such as diabetes, hypertension and endocrine insufficiencies.
The etiology for diabetes is known to be mostly owing to disturbances of insulin secretion in the pancreatic xcex2-cells. Consequently, elucidation of the molecular mechanism of insulin secretion is expected to play an important role in the clarification of causes for diabetes and the development of therapeutic agents against diabetes, but no detail has yet been made known on such molecular mechanism.
It has already been made clear that the ATP-sensitive potassium channel (KATP) being present on the cellular membrane plays a leading role in the cellular functions such as secretions and muscular contraction by coupling the state of metabolism in the cells with the membrane potential.
The KATP channel was first discovered in the cardiac muscle in 1983 [Noma, A., Nature 305:147 (1983)] and was thereafter confirmed to be present in tissues such as the pancreatic xcex2-cell [Cook, D. L. et al., Nature 311: 271 (1984), Misler, S. et al., Proc. Natl. Acad. Sci. U.S.A. 83: 7119 (1986)], pituitary [Bernardi, H. et al., Proc. Natl. Acad. Sci. U.S.A., 90:1340 (1993)]. skeletal muscle [Spruce, A. E., et al., Nature, 316: 736 (1985)] and brain.
In addition, it has been suggested that there exists the molecular heterogeneity of such KATP channels [Ashcroft, F. M., Annu. Rev. Neurosci. 11: 97 (1988)].
Particularly in the pancreatic xcex2-cells, ATP produced by the metabolism of glucose brings about calcium ion inflow from the calcium channel by closing the KATP channel to cause depolarization, resulting in secretion of insulin. As is evident from this, the KATP channel plays a leading role in regulating the secretion of insulin.
The KATP channel belongs to a potassium channel family exhibiting electrophysiologically inward rectification, whereby the potassium channel family exhibiting inward rectification is classified into the four subfamilies, ROMK1, IRK1, GIRK1 and cKATP-1, on the basis of the degree of amino acid sequence identity.
Nevertheless, there has not been clarified the molecular architecture for the KATP channel in the pancreatic xcex2-cells. In addition, no information has been disclosed on the novel ATP-sensitive potassium channels (huKATP-1 and ruKATP-1) of the present invention for the detailed protein structure and the formation of complexes with other proteins, for example, the sulfonylurea binding protein.
In order to achieve the isolation, identification and functional analyses of a novel membrane channel, there are required the sophisticated techniques, such as molecular biological technique, cellular biological technique and electro-physiological technique.
Such being the case, the present inventors made ample and full use of such techniques to isolate human and rat genomes and cDNAs encoding the novel KATP channel (uKATP-1) expressed in different tissues of mammalians and to identify their amino acid sequences (see FIGS. 1, 2, 3 and 4). The identified uKATP-1 channel was expressed in the Xenopus oocyte system and mammalian cell lines.
Electrophysiological analysis demonstrated that uKATP-1 is an ATP-sensitive potassium channel exhibiting inward rectification. The uKATP-1 channel being expressed ubiquitously in tissues of mammalians inclusive of man and rats is involved in the maintenance of the membrane potential through the basal energy metabolism.
As is described in the above, the present invention relates to an ATP-sensitive potassium channel (uKATP-1) which is ubiquitously present in mammalians, and encompasses the ATP-sensitive potassium channel proteins, identified DNA sequences encoding the same, plasmid having such sequences incorporated therein and furthermore recombinant cells (tranformants) having such plasmid transfected therein. In addition, this invention comprises the isolated UKATP-1 proteins and recombinant proteins, their related materials such as agonists and antagonists, and drug designs inclusive of diagnostics and drugs for gene therapy.
huKATP-1 of a human origin is composed of 424 amino acid residue (See FIG. 1 (SEQ ID NO: 1)) with a molecular weight of 47,965, while the one of a rat origin is likewise composed of 424 amino acid residue (see FIG. 4 (SEQ ID NO: 4)) with a molecular weight of 47,960. These two potassium channels exhibit 98% amino acid sequence identity, and such a marked homology leads us to the assumption that uKATP-1 performs common, structurally and functionally basic actions in all mammalian cells. Among others, uKATP-1 participates in the membrane potential and energy metabolism, suggesting that it could find application as a drug substance acting to prevent disturbances under unusual, extreme metabolic conditions inclusive of endocrine diseases, e.g. diabetes, starvation and ischemia.
For example, the inflow and outflow of calcium ions caused by the opening and closing of UKATP-1 during the onset of ischemia is closely connected with ischemic disturbances. In other words, there is a possibility that the agonists and antagonists for the opening and closing of uKATP-1 would constitute a suppressory agent against ischemic disturbances.
From the comparative studies of huKATP-1 and ruKATP-1 with other potassium channels for the amino acid sequence, it was confirmed that uKATP-1 of the present invention belongs to a novel family of the inward rectifier potassium channels; the central region of the uKATP-1 protein showed incresed homology with other inward rectifier potassium channels. A hydropathy plot indicated the presence of two hydrophobic regions, which are composed of two transmembrane regions characteristic of the inward rectifier potassium channels and one pore region [Nicholas, C. G., Trends Pharmacol. Sci., 14: 320 (1993), Jan, L. Y. and Jan, Y. N., Nature, 371: 119 (1994)].
With reference to ruKATP-1 (Inagaki, N. et al., J. B. C., 270: 5691 (1995)], it was reported that in the second intracellular region, there are two potential cAMP-dependent protein kinase phosphorylation sites (Thr-234 and Ser-385) and seven potential protein kinase C dependent phosphorylation sites (Ser-224, Thr-345, Ser-354, Ser-379, Ser-385, Ser-391 and Ser-397), while there are one (Thr-63) and four potential casein kinase II dependent phosphorylation sites (Thr-234, Ser-281, Thr-329 and Ser-354) in the first and second intracellular regions, respectively, with no N-linked glycosylation site being present in the intracellular regions. The same findings were obtained with huKATP-1 [Inagaki, N., et al., in press (1995)].
Then, the present inventors identified the nucleotide sequences and entire amino acid sequences of huKATP-1 and ruKATP-1, thus enabling not only proteins themselves of huKATP-1 and ruKATP-1 but also their mutants to be synthesized in large quantities by expressing the DNAs encoding huKATP-1 and ruKATP-1 and their mutants in bacteria or animal cells with use of the known genetic engineering techniques. It is furthermore added that huKATP-1 and its fragments are useful for the hybridization diagnosis of depleted huKATP-1 DNA, with the mutants of huKATP-1 being of use in the studies on the sugar metabolism in cells, particularly insulin-dependent and independent diabetes.
The DNAs of novel huKATP-1 and ruKATP-1 according to the present invention were identified based on a cDNA library and genome library. The DNA encoding huKATP-1 shows a length of about 9.7 kb, being composed of three exons and is present on the chromosome at 12p11.23. The chromosomal DNA can be obtained by probing a genome DNA library with use of cDNAs for uKATP-1 and its fragment, as well. The isolated uKATP-1 DNA can easily be subjected to nucleotide depletion, insertion or replacement by the known techniques to prepare its mutants.
By employing the known techniques, it is easy to link nucleotide sequences encoding other proteins or synthetic polypeptides to uKATP-1 or its variants at the 5xe2x80x2 and 3xe2x80x2 ends to thereby prepare fusion proteins, or derivatives thereof.
For example, a fusion protein is prepared as a precursor protein and undergoes cleavage in vivo or in vitro to thereby perform functions; such fusion protein provides target-tissue and membrane orientation in addition to its proper function. In such a case, the fusion proteins contain sugar-chain binding amino acids, and can be modified to derivatives having tissue orientation or physiological activities activated by adding new sugar chains.
In order to produce uKATP-1, its mutants or their derivatives, the corresponding coding DNA is incorporated into a reproducible plasmid, and host cells being transformed with such plasmid are incubated. The host cells include bacteria, yeasts and animal cells.
Prokaryotes such as bacteria are suited for the cloning of deoxyribonucleotides. For example, pER 322 plasmid derived from E. coli contains a gene resistant to ampicillin or tetracycline and can provide a practical means of identifying the transformed cells. Furthermore, the microbial plasmids contain a promoter which can be used to express their proteins themselves. In addition to prokaryotes, eukaryotes such as yeasts can work well, with a plasmid YRp7 being utilizable especially in allowing the expression in yeasts of the species Saccharomyces [Stinchomb et al., Nature, 282: 39 (1979)].
Animal cells are also used as a host, and particularly the incubation of vertebra cells is employable easily and constitutes a conventional means [Krause and Paterson, Tissue Culture, Academic Press (1973)]. As the cell lines, there are mentioned AtT-20, Hela cells, Chinese hamster ovary (CHO), COSM6, COS-7 and the like. The promoters of Polyomavirus, Adenovirus 2, Cytomegalovirus and Simian virus 40 are used to control the function of expression plasmid in such cell lines, wherein pCMV is a plasmid which finds widened application in the expression systems of animal cells [Thomsen et al., PNAS, 81: 659 (1984)].
The DNA sequences for the channel protein and huKATP-1 and ruKATP-1 according to the present invention begin with the initiation codon xe2x80x9cATGxe2x80x9d. In cases where the recombinant cells are used to synthesize such protein, there is no need to add ATG to the desired DNA, thus making the manipulation easy. When uKATP-1 is expressed in a prokaryote transformed with E. coli, consequently, there is generally synthesized a protein of the amino acid sequence beginning with Met. The N-terminated met of the resultant protein may be eliminated according to the purpose of application.
In cases in which uKATP-1 is synthesized in recombinant animal cells, similarly, proteins having Met contained or eliminated at the N-terminal are bio-synthesized, and both are useful for individually intended application purposes.
uKATP-1 and its fragments can be administered to animals for their immunization to thereby produce antibodies. Also, immunization of animals permits a monoclonal antibody to be produced from cells secreting the desired antibody.
It has become easy to prepare uKATP-1 in large quantities, thus providing better understanding of the same at the molecular level. Accordingly, the production of uKATP-1 and its mutants or analogs raises the possibility to develop diagnostics or therapeutics for the channel-protein related diseases.
In particular, such proteins can be utilized in the procedures of investigating into a substance suited for diagnostics and therapeutics, or a substance that exerts agonistic or antagonistic action on uKATP-1. For example, a testing procedure with animal cells can be conducted by injecting cRNA for uKATP-1 into cells to conduct expression, followed by addition of sulfonylurea to study their interactions [Kayano, T. et al., J. Biol. Chem., 265: 13276 (1990), Example 4].
Additionally, the pertinent information has been obtained on the DNA sequence of uKATP-1, facilitating DNA or RNA encoding their fractional sequences to be prepared. Such relatively short DNA sequences possess the capability to hybridize with the gene to be selected, and can find application as a probe, which probe is effective for detection of cDNAs in different tissues.
The probe as prepared with use of uKATP-1 can be utilized to produce nucleic acids capable of hybridization from a variety of organisms and their tissues. The resultant nucleic acids may be the same type as uKATP-1 or its isoform and include nucleic acids encoding the novel proteins.
The prepared probe is utilizable in the gene diagnosis of potassium-channel related diseases; investigation can be conducted into patients"" nucleotide sequences hybridized with the probe capable of detecting the disease genes.
The blocker and opener agents for the potassium channel have heretofore been used as therapeutics against diabetes and hypertension. uKATP-1 and its mutants, their derivatives and monoclonal antibodies to them, when processed into pharmaceutical preparations, can be administered to patients to thereby alleviate through neutralization the adverse effects brought about by an excess of such blocker or opener agents administered clinically. When uKATP-1 itself shows functional insufficiency, such pharmaceutical preparations can be administered to thereby make up for such deficient functions of uKATP-1.
The present invention comprises the preparation of drugs for gene therapy being applicable in the essential treatment method. The nucleotide sequences for uKATP-1 or its mutants and their derivatives can be incorporated into plasmid or stem cells, which are then given patients to open up the possibility of finding application as a drug for gene therapy.
Below described are the examples to illustrate the present invention in more detail, while referring to the appended drawings.